Novel Zoonotic Avian Influenza A(H3N8) Virus in Chicken, Hong Kong, China

Zoonotic and pandemic influenza continue to pose threats to global public health. Pandemics arise when novel influenza A viruses, derived in whole or in part from animal or avian influenza viruses, adapt to transmit efficiently in a human population that has little population immunity to contain its onward transmission. Viruses of previous pandemic concern, such as influenza A(H7N9), arose from influenza A(H9N2) viruses established in domestic poultry acquiring a hemagglutinin and neuraminidase from influenza A viruses of aquatic waterfowl. We report a novel influenza A(H3N8) virus in chicken that has emerged in a similar manner and that has been recently reported to cause zoonotic disease. Although they are H3 subtype, these avian viruses are antigenically distant from contemporary human influenza A(H3N2) viruses, and there is little cross-reactive immunity in the human population. It is essential to heighten surveillance for these avian A(H3N8) viruses in poultry and in humans.

The Department of Agriculture, Fisheries and Conservation in Hong Kong routinely conducts virologic surveillance on each batch of chickens from local farms before release for sale. The surveillance is conducted on 30 unvaccinated sentinel chickens cohoused with each chicken flock. During December 14, 2021-January 21, 2022, we obtained oropharyngeal and cloacal swab samples from 30 chickens on each of 28 poultry farms. We combined samples into pools of 6 and placed each pool into a vial of virus transport medium (medium 199 plus antimicrobial drugs).
In a follow-up investigation of 4 farms found positive for H3N8 virus, we conducted more intensive Zoonotic and pandemic influenza continue to pose threats to global public health. Pandemics arise when novel influenza A viruses, derived in whole or in part from animal or avian influenza viruses, adapt to transmit efficiently in a human population that has little population immunity to contain its onward transmission. Viruses of previous pandemic concern, such as influenza A(H7N9), arose from influenza A(H9N2) viruses established in domestic poultry acquiring a hemagglutinin and neuraminidase from influenza A viruses of aquatic waterfowl. We report a novel influenza A(H3N8) virus in chicken that has emerged in a similar manner and that has been recently reported to cause zoonotic disease. Although they are H3 subtype, these avian viruses are antigenically distant from contemporary human influenza A(H3N2) viruses, and there is little cross-reactive immunity in the human population. It is essential to heighten surveillance for these avian A(H3N8) viruses in poultry and in humans.
surveillance during May 2022 to check for any continuing evidence of on-farm viral circulation. We sampled a total of 50 chickens by using oropharyngeal and cloacal swabbing, again in pools containing 6 specimens.

Influenza A Surveillance in Live Poultry Markets
The School of Public Health of The University of Hong Kong routinely conducts surveillance in live poultry market stalls in Hong Kong (n = 116) by sampling from each stall fecal droppings (n = 10), drinking water in poultry cages (n = 2), and chopping boards and the inner wall and outer surface of defeathering machines used (n = 3) in preparation of slaughtered poultry for sale (7). All 116 poultry stalls were sampled every 3 months. Samples were individually collected and placed into vials of virus transport medium (medium 199 plus antimicrobial drugs), and samples were kept in cool packs for transport to the laboratory.

Real-Time Reverse Transcription PCR for Detection of Influenza A Viruses
We extracted viral RNA from chicken oropharyngeal and cloacal swab specimens by using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche, https://lifescience.roche.com) according to the manufacturer's instructions. We tested eluted specimen RNA by using real-time reverse transcription PCR (RT-PCR) for the influenza A virus matrix (M) gene as described (8).
We tested swab specimen supernatants of all influenza A virus M gene-positive swab specimens by RT-PCR for H5, H7, and H9 (9) and for virus isolation. We identified virus subtype of M gene-positive swab specimens negative for H5, H7, and H9 by using genetic sequencing of the virus isolate or directly from the swab specimen.

Virus Isolation
We inoculated 0.2 mL of swab specimen supernatant of all influenza A virus M gene-positive swab specimens into the allantoic cavity of three 9-11-day-old specific pathogen-free embryonated eggs and incubated at them at 36°C (± 2°C) for 4 days. We candled the eggs daily, and harvested allantoic fluid. We subtyped virus isolates by using hemagglutination inhibition (HI) tests and reference panels of antiserum to a range of influenza virus A subtypes (7).

DNA Bar Coding
We conducted PCR amplification of the mitochondrial cytochrome oxidase I gene for host-species-   identification as described (18). We sequenced the amplified ≈700-bp PCR fragment of the cytochrome oxidase I gene by using the 3730xl DNA Analyzer (Applied Biosystems, https://www.thermofisher. com) and analyzed by using the barcoding software bold, which provides a taxonomic assignment to the query sequence by using a linear search to collect nearest neighbors (lowest percentage divergence) from a global alignment of all reference sequences (19).

Serologic Analysis
We used the HI test to detect the seroprevalence to 1 of the novel H3N8 viruses, A/chicken/Hong Kong/ MKT-AB13cp/2022, and human seasonal virus A/ Switzerland/8060/2017 (H3N2) in a panel of agestratified blood donor serum samples collected during 2019-2020. The study protocol was approved by the University of Hong Kong. We also tested HI titers of a World Health Organization reference antiserum to A/Switzerland/8060/2017 against A/chicken/ MKT-AB13cp/2020 H3N8 virus (original serum dilution provided was 1:128) in comparison with the homologous virus A/Switzerland/8060/2017. The HI tests were conducted as described (20,21). We analyzed the effect of age-stratified seroprevalence on the reproduction number (R 0 ) and population immunity as described (22).

Results
During routine virologic surveillance on chicken farms, H3N8 viruses were first identified on samples collected from 2 broiler farms (farms A and B) in December 2021 and subsequently detected on 2 other broiler farms in January 2022 (farms C and D) (  Table 2). Chicken producers were subsequently advised to conduct thorough disinfection and strengthen farm biosecurity to prevent further spread and eliminate the virus.
Follow-up virologic testing in May 2022 of 150 chickens from each of the 4 positive farms yielded negative results ( Table 3). As of the end of June 2022, there has been no additional detection of H3N8 on any farms.
During January 2022-June 2022, we collected and tested 3,525 environmental swab samples of fecal droppings, drinking water in poultry cages, and chopping boards and defeathering machines in live poultry markets and stalls sampled (Table 4). An environmental swab specimen collected from a chicken defeathering machine on January 12 and a swab specimen collected from a poultry chopping board on January 20 from 2 different live poultry markets were positive for influenza A(H3N8) viruses. The second market only sells chicken, and the first market additionally sells chilled dressed duck slaughtered elsewhere. The species of origin from both swab specimens was determined by DNA bar coding to be domestic chicken (Gallus domesticus).
We sequenced 7 H3N8 viruses in this study and submitted them to GISAID (      Table 5 were analyzed with other relevant virus sequence data available in public databases (accession numbers in Appendix Table,  each other and to an H3N8 virus associated with zoonotic disease in mainland China (Figure; Appendix Figure). The polymerase basic 1, polymerase basic 2, polymerase acidic, NA, nonstructural protein, and M gene segments were derived from the G57 sublineage of influenza A(H9N2) viruses commonly found in mainland China (23), whereas the HA gene sequences belong to the Eurasian avian H3 lineage, which has been detected in ducks and other wild birds (24).
The NA gene sequences of the poultry A(H3N8) viruses belonged to the North American lineage, but a closely related N8 NA sequence had previously been detected in A/Env/Hong_Kong/MP16_1834/2016 (H3N8), a virus isolated on December 21, 2018, from the Mai Po Wetlands, Hong Kong, in 2018, obtained from a fecal specimen identified by DNA bar coding to be derived from a Northern pintail duck (Anas acuta) ( Table 5). Two other H3N8 viruses isolated from fecal droppings collected from the Mai Po Wetlands on November 14, 2018, identified to be from a Northern shoveler duck (Anus clypeata) were genetically unrelated in all gene segments to the chicken H3N8 viruses. The N8 gene segment sequence also is closely related to other aquatic wild bird H3 viruses from mainland China. Other than for the N8 NA gene segment, none of the other gene segments of the poultry H3N8 viruses were derived from the wild bird H3N8 viruses detected in the Mai Po Wetlands of Hong Kong. These viruses were distinct from chicken H3N8 viruses previously reported in mainland China (25). However, 1 sequence of a virus from chicken similar in all 8 gene segments to our Hong Kong H3N8 viruses is available in virus genetic sequence databases (Figure).
The HI titer of the World Health Organization reference antiserum to human seasonal H3N2 virus A/Switzerland/8060/2017 against the homologous virus antigen was 1:128, and the titer against A/ chicken/Hong Kong/MKT-AB13cp/2022 was <1:10, suggesting limited antigenic cross-reactivity of current human seasonal H3N2 viruses with these novel avian H3N8 viruses. The overall seroprevalence (HI titer >1:40) to A/chicken/Hong Kong/MKT-AB-13cp/2022 (H3N8) in age-stratified human serum samples was 3.2% (Table 6). In contrast, as expected, we found high (58.7%) seroprevalence to a recent human seasonal A/Switzerland/8060/2017 (H3N2) virus in this same panel of serum samples.
Human population immunity to a potentially zoonotic virus is a major parameter that is included in the risk assessment of animal viruses for a pandemic threat. We have described an approach to assess that risk by estimating the effect of age-stratified immunity in the human population by using HI tests on R 0 of such a virus if it were to become transmissible in humans (22). We found that the observed seroprevalence in humans would provide little or no resistance to such a virus, if it were to acquire other factors required for transmission between humans (Table 7).

Discussion
We report detection of chicken influenza A(H3N8) viruses from live poultry markets and farms in Hong Kong. These viruses were genetically similar to each other and to a recently reported zoonotic H3N8 virus in mainland China (6). The viruses were novel reassortants that have virus internal gene segments   Table). These H3N8 viruses were also distinct from H3N8 viruses reported in horses, dogs and cats (27)(28)(29). These novel H3N8 viruses appear to have arisen in a manner analogous to the emergence of previous zoonotic H7N9 and H10N8 viruses, in which the H9N2 viruses enzootic in chicken and other game birds in China acquired HA and NA gene segments from wild, aquatic bird viruses. Wild aquatic birds share ecosystems with domestic ducks, and it is inevitable that influenza viruses will also be shared in such ecosystems. Subsequent trade systems in which domestic ducks and chickens (and other game birds) are mixed in close proximity within wholesale and retail poultry markets provide the opportunity for H9N2 viruses in chicken to acquire HA and NA gene segments from domestic ducks, as has been postulated in the emergence of H7N9 and H10N8 viruses (4).
Pandemics emerge when influenza viruses of birds, swine, or other mammals adapt to transmission between humans and when the human population lacks immunity to the hemagglutinin of the newly emerged virus. Cross-reactive immunity in humans is 1 parameter that is considered when risk assessing the pandemic threat from a newly emerged animal influenza virus (30). Our data suggest that there is little antigenic cross-reactivity between contemporary seasonal H3N2 viruses and the H3N8 virus. The overall HI test seroprevalence at a titer >1:40 to H3N8 in age-stratified serum samples collected from blood donors in Hong Kong was 3.2%, and the estimated proportion of the population immune (weighted for age structure) was 2.9% (95% CI 1.2%-5.8%). We estimated that if this H3N8 virus acquired transmissibility between humans and acquired an R 0 >1.033, cross-reactive population immunity would fail to impede its onward transmission in the human population. For comparison, similar estimation of the minimal R 0 required for the 2009 pandemic H1N1 virus to spread in face of population immunity before its emergence and spread in 2009 was 1.231 (95% CI 1.185-1.292), a markedly higher threshold to cross (22).
In conclusion, we report the emergence of a novel influenza A(H3N8) virus in chickens in Hong Kong. This virus might have major zoonotic and pandemic potential. Our results indicate the need to enhance surveillance for this virus in poultry, carry out comprehensive risk assessment of such a virus, and prepare pandemic seed vaccine strains if justified by such risk assessment.